The present invention relates to novel adhesive mucilage isolated from the fungus Mggnaporthe grisea, and to procedures for such isolation.
Many biological organisms must attach to a surface for survival. This attachment often occurs in an aqueous habitat. This is an interesting phenomenon since adhesion in aqueous environments is difficult because adhesives are generally adversely affected by the presence of water on the substrates being adhered. Water competes with the adhesive for the surface, tends to hydrolyze the adhesive, and frequently plasticizes it. Accordingly, it is usually required that the substrate surfaces being adhered be substantially free from water or other aqueous impurities. As can be appreciated, such conditions are not always possible, particularly for bioadhesives used in medical and dental applications and employing a wide variety of substrates such as those encountered when gluing or restoring fractured hard tissue in the body such as bone, cartilage and teeth, as well as ligaments, blood vessels and the like.
It would be useful if natural bioadhesives could be routinely isolated and adapted for industrial applications. Unfortunately, this is not the case. Attempts to isolate them generally would be expected to be attended by significant difficulties due to the sticky nature of the material. In addition, one of skill in the art would expect that purification attempts might result in loss or decrease of the adhesive nature of the material, due to one or more of the following:
1) Bioadhesives are generally polysaccharides, glycoproteins, proteoglycans or lipopolysaccharides. Therefore, they would be expected to be subject to degradation during isolation by, for example, proteases, glycosidases, and/or lipases.
2) Some bioadhesives are known to have chemically reactive groups which may undergo undesirable modification during purification if precautions are not taken. A specific example of this is the mussel adhesive isolated by Waite (U.S. Pat. No. 4,496,397), in which there are numerous o-diphenyl groups which can undergo autooxidation, with resultant loss of adhesive activity.
3) Some known bioadhesives are multicomponent in nature. Thus purification of any one part would result in less than optimal adhesive properties of the isolated material when compared with the natural, multicomponent material. For example, the mussel adhesive noted above contains ". . . a polyphenolic substance that is mixed by the animal's foot with a curing enzyme (phenoloxidase) and a mucosubstance to provide a complex three-component natural adhesive system". In addition, the interconnective tissue of animal cells often involves supramolecular complexes of various proteoglycans (Rollins, B. J., et al., "Fibronectin-proteoglycan Binding as the Molecular Basis for Fibroblast Adhesion to Extracellular Matrices". In The Glycoconjugates, Academic Press (1982)).
4) Any dispersion and reformation of a (non-covalently linked) supramolecular complex with adhesive properties runs the risk of incorporating (trapping) material that originally was not part of the adhesive structure. Any decrease in the interconnecting bonds of an adhesive would decrease the adhesive strength of the material. Moreover, contaminants may bind to the adhesive portion of the structure, thereby reducing the adhesive ability of the structure.
5) Many instances of bioadhesion are thought to rely solely upon the forces of capillarity. In these instances the viscosity of the fluid that occupies the capillary spaces will play an important and direct role in the strength of these capillary forces: the higher the viscosity the greater strength of the adhesion. Based upon these facts, it is reasonable to believe that a reduction or elimination of capillary fluid viscosity, during the course of isolation of the adhesive material as would occur upon such treatments as would cause dispersion of a mucilage, would correspondingly reduce or eliminate the adhesive properties of that fluid.
Among fungi which must adhere in aqueous environments, marine fungi adhere to underwater surfaces via mucilages and appendages (Rees, G. and Jones, E. B. G., Botanica Marina 27:145, 1984), human fungal pathogens adhere to the cell surfaces of the invaded areas via extracellular polysaccharides (McCourty, J. and Douglas, L. J., J. Gen. Microbiol., 131:495, 1984), and fungal plant pathogens adhere to wet plant surfaces via mucilages (Hamer, J. E., et al., Science 239:288, 1988). Despite these observations of fungal adhesion, only rarely has the origin of the adhesion been experimentally demonstrated (Rees, supra). Even less is known about the biochemical nature of fungal mucilages (Ramadoss, C. S., et al., J. Ag. and Food Chem., 33:728, 1985). Although the adhesive nature of fungal mucilages has been postulated, in no case has a fungal mucilage been isolated from the organism, purified, characterized and demonstrated to retain useful adhesive properties. No attempts have been reported, presumably due to the general expectation of difficulties disclosed above.
The fungus Magnaporthe grisea is found worldwide as a pathogen of many grasses, where it attacks the aerial parts of the plant. Common synonyms for M. grisea include Pyricularia grisea and Pyricularia oryzae. The fungus can easily be isolated in pure culture form from lesions on infected plants. Asexual spores (conidia) are dispersed to plant surfaces by wind or rain. During warm, humid conditions, these conidia germinate and infect the plant. To infect the plant, however, the conidia must attach to the plant surface. For Magnaporthe grisea, this attachment occurs at the conidial tip. Flow chamber studies have shown that conidia adhere tightly by their tips, and that concanavalin A blocks conidial attachment. It has been hypothesized that mucilage might be the agent of attachment (Hamer, supra).
The term mucilage has a generic meaning, and includes many compositions which are, by definition, sticky. The common mucilage used for gluing paper, for example, is comprised of complex plant polysaccharides. This is very different from the polyphenolic mucilage protein obtained from mussel (U.S. Pat. No. 4,496,397), which was identified as a polyphenolic protein rich in 3,4-dihydroxyphenylalanine (dopa) and hydroxyproline. This protein was disclosed as being very difficult to isolate, and the actual bioadhesive is a complex of three distinct components. In particular, it is noted that U.S. Pat. No. 4,496,397 is directed to a process for purifying and stabilizing the catechol-containing polyphenolic protein portion of the bioadhesive, which, as noted above, involves procedures outside of the usual and expected means usually used to isolate and purify "normal" nonmucilagenous proteins.
It is also interesting to note that yeasts which stick to plants have been shown to produce novel extracellular lipids, including a glucose-containing disaccharide linked to lipid. This has been shown to be a glucose disaccharide in which a terminal glucose is beta-linked to the 2-position of another glucose residue. This disaccharide is glycosidically linked to 13-hydroxydocosanoic acid. These yeast lipids have not been shown to be involved in adhesion. See "Extracellular Lipids of Yeasts" (Stodola et al., Bacteriological Reviews, Sept. 1967, p. 194-213).
Research on the biochemical nature of the fungal adhesives is particularly difficult due to extremely small amounts of mucilage present in each spore tip, the contamination of extracellular polysaccharides which are secreted when the fungal mycelia are grown in culture and the physical characteristics of the adhesive itself. In particular, it is to be noted that mucilages in natural form, especially in concentrated form, are viscous, sticky substances which adhere to even hydrophobic surfaces, making manipulation and transfer of the substance difficult and losses high at each step. Thus, in view of the particular problems expected to be encountered with fungal spore tip mucilage as well as the general problems encountered with other bioadhesives, it can be understood that isolation and purification of spore tip mucilage in useful quantities was assumed to be difficult and unrewarding, especially in view of the very significant possibility that the material which would be obtained would not retain its adhesive properties. In fact, the inventors of the instant invention made many unsuccessful attempts to purify fungal mucilage prior to the development of the instant invention. In particular, numerous forms of column chromatography were attempted, but great difficulty was encountered in removing the material from the column matrices.